In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

Abstract

NAD is an essential metabolite that exists in NAD + or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD + /NADH redox state and modulating cellular signaling processes through the activity of the NAD + -dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD + and NADH contents and the NAD + /NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD + , total NAD contents, and NAD + /NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.redox state | NAD | in vivo 31 P MR spectroscopy | human brain | aging N AD, a multifunctional metabolite found in all living cells, has been the interest of many scientific investigations since its discovery in the early 20th century (1). NAD is known to convert between its oxidized NAD + and reduced NADH forms during the breakdown of nutrients; hence, the intracellular NAD + /NADH redox state reflects the metabolic balance of the cell in generating ATP energy through oxidative phosphorylation in mitochondria and/or glycolysis in cytosol (2). More recently, after several protein families associated with cell survival were found to use NAD + as their main substrate with activities also regulated by the availability of the NAD + , the full extent of the NAD's function as a metabolic regulator began to unfold (3-5). A growing number of studies have indicated that NAD + can modulate metabolic signaling pathways and mediate important cellular processes, including calcium homeostasis, gene expression, aging, degeneration, and cell death; therefore, the cellular NAD could serve as a therapeutic target for treating various metabolic or age-related diseases and promoting longevity (6-12).Despite the critical relevance of the intracellular NAD metabolism to human health and diseases, assessment of NAD contents and NAD + /NADH redox state is extremely challenging. Only a few invasive techniques based on biochemical assays or autofluorescence methods have been used to analyze tissue samples or cell extracts (13,14). However, during the preparation of such ex vivo sample, the NAD + and NADH contents are likely altered, beca...

Redox Dysregulation in Schizophrenia Revealed by in vivo NAD+/NADH Measurement

“…29 NADH and RR for SZ and healthy participants are plotted against age in figures 4a and 4b, demonstrating replication of RR decline in healthy participants 29 and an extension of this finding to SZ patients. In addition, the younger participants have lower RR than older ones, apparently reversing the age-dependent trend.…”

“…Thus, RR is closely linked with brain carbohydrate metabolism and the balance between glycolysis and oxidative phosphorylation. 29 Mitochondrial dysfunction can also contribute to abnormal RR independent of glycolysis. The regeneration of NAD+ in the ETC takes place in particular at the mitochondrial complex I.…”

“…Recently, a 31 P MRS-based NAD quantification method has been developed to directly quantify NAD+ and NADH concentrations in vivo at ultra-high field MR scanners (7.0 and 9.4 Tesla). 28,29 This method applied at a 4 Tesla (4 T) scanner was recently reported. 30 This approach is based on a theoretical NMR model with prior knowledge (ie, chemical shift and J-coupling constants) to predict phosphorus NAD+ and NADH resonance signals.…”

“…In order to remain consistent with and compare to prior reports, the concentrations of NAD+ and NADH were determined using α-ATP signal as an internal reference, in which its brain concentration was set to 2.8 mM. 28,29 For the validity of our measurement, we summed all spectra of individual participant in group of chronic SZ and matched controls and the summed spectra were fitted again. In this case, uridine diphosphate glucose (UDPG) was also included in the fitting routine with published values of chemical shift and coupling constant 31 to indentify the potential contaminations of UDPG to NAD content.…”

Mitochondria and aging: A role for the mitochondrial transition pore?

“…This was observed in several organs in mice and in Caenorhabditis elegans . There is also evidence of NAD + reduction in aged human tissues (Fang et al., 2014; Massudi et al., 2012; Mills et al., 2016; Mouchiroud et al., 2013; Zhang et al., 2016; Zhu, Lu, Lee, Ugurbil, & Chen, 2015). Conversely, supplementation with NAD + precursors or overexpression of a NAD + synthetic enzyme nicotinamidase have been reported to extend lifespan and to improve healthspan in different species (for review, see Fang et al., 2017).…”